ABSTRACT Progress in cellular reprogramming technologies has created alternative platforms for scalable production of blood cells for transfusion, immunotherapies and transplantation through inducing pluripotency in somatic cells. However, even with advances hematopoietic differentiation methods, primitive wave of hematopoiesis dominates pluripotent stem cell (PSC) differentiation cultures and markers that distinguish primitive and definitive lymphomyeloid hematopoiesis remains largely unknown. Thus, further translation of hPSCs to hematology clinic requires a better understanding of the molecular program guiding definitive lymphomyeloid hematopoiesis. During development, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. The lack of venous contribution to HSCs along with the common signaling pathways required for both arterial fate acquisition and HSC development, led to the hypothesis that arterial specification is a critical prerequisite for HSC formation. However, a direct progenitor- progeny link between arterial endothelium and definitive lymphomyeloid hematopoiesis has never been demonstrated. In present application, we propose to prove the hypothesis that arterial specification is an essential prerequisite for definitive hematopoiesis and demonstrate that promotion of arterial patterning of HE can provide a novel strategy to aid in generating of lymphoid cells from hPSCs for immunotherapies. In aim 1, we will identify arterial type of HE (AHE) and demonstrate a direct progenitor-progeny link between AHE and definitive lymphomyeloid hematopoiesis using arterial-specific enhancer-Cre tracing system. In aim 2, we will demonstrate that arterial program activation is essential for establishing definitive lymphomyeloid hematopoietic program. We will show that enhancement of definitive hematopoietic program from hPSCs can be achieved through activation of arterial program with arteriogenic ETS and SOXF transcription factors (TF), and modulation of the molecular pathways involved in arteriogenesis using small molecules. In contrast, we will show that inhibiting arterialization following HE specification abrogates definitive hematopoiesis. Using RNAseq and ChipSeq analysis we will identify a gene regulatory network connecting arterial and definitive hematopoietic programs. In aim 3, based on the knowledge gained in understanding the role of arteriogenic factors in lymphopoiesis, we will develop a forward programming system for T cell generation from hPSCs using modified mRNA and assess their suitability for CAR-T cell therapies in vivo. Overall, the proposed studies will establish for the first time a molecular link between arterial programming and definitive hematopoiesis, and provide evidence that promoting arterial patterning in hPSC cultures can aid to in vitro approaches to instruct definitive hematopoiesis with lymphoid potentials from hPSCs. In addition, we will offer a novel system allowing for scalable off-the-shelf production of T cells from hPSCs for immunotherapies.